The need for efficacious mucosal vaccines has received considerably increased attention due to: 1) the rise in antibiotic resistant enteric pathogens and the threat of gram-negative bacterial sepsis; 2) the unexpected cholera epidemics in South America; 3) the prominence of bacterial diarrhea as a killer of infants; 4) the increased incidence of tuberculosis caused by antibiotic-resistant variants; and 5) the continued spread of HIV infections. Because of our still incomplete understanding of certain major features of the mucosal immune response, attempts to develop new mucosal vaccines are still largely empiric. We plan to continue our productive program to understand the mechanisms leading to a mucosal immune response by: 1) trying to elucidate, at cellular and molecular genetic levels, the role of Peyer's patch (PP) (aggregates of lymphoid follicles in the intestinal walls) germinal centers in the development of a humoral mucosal immune response; 2) defining the cell/cell and cell/cytokine exchanges that account for the effectiveness of our B- and T-lymphocyte plus dendritic cell cultures at generating an lgA antibody response; 3) evaluating the contributions of the B2 (follicular) vs. systemic B1 subsets of B cells to protective mucosal immune responses; and 4) analyzing mechanisms that regulate the host's mucosal immune response to enteric gram-negative bacteria and facultative, intracellular gram-positive pathogens, such as Listeria, infecting via the mucosal route. We plan to use germ-free and antigen (Ag)-free mice, which have been very informative for analyzing acute, de novo gut mucosal responses, and a new generation of in vitro functional assays which we have recently developed: 1) the PP and lamina propria tissue fragment assay, which reports the immune status of the gut at the time of tissue sampling; and 2) the single/clonal B cell microculture, which reveals the lg isotype potential, frequency, and physiologic state of Ag-specific B cells. These should permit us to address our aims, for instance by permitting us to quantitate the temporal fluctuations and whereabouts of Ag-specific lgA-memory cells, IgA-pre-plasmablasts, and lgA plasma cells following acute oral stimulation with enteric viruses, bacteria, or toxins. Cross regulation and stimulation of systemic vs. mucosal compartments can also be examined in our system. Finally, we plan to probe how a mucosal response to a novel Ag, given in the context of copious amounts of a wide array of environmental Ags, may differ from the better understood systemic immune response.